For many years, medical diagnostic tools have used classical biochemical techniques that involve bulky and expensive equipment such as spectrophotometry, gas chromatography (GC), mass spectrometry (MS), high-performance liquid chromatography (HPLC), paper and thin-layer chromatography (PC and TLC), and electrophoretic techniques coupled with fluorescence detection techniques. These standard analytical tools work effectively and efficiently. However, the tools are expensive, and they require costly consumables, sample throughput, and experienced and skilled operators. All these hinder rapid, inexpensive, and in-situ diagnosis of health-care requirements. Furthermore, such methods often require tedious and laborious processes. For these reasons, these tools are mostly used as confirmatory tools for the presumptive positive samples that are initially screened by some types of assay techniques.
Currently, the problems with quantitative immunoassay techniques are not significantly different from the classical biochemical techniques mentioned earlier. The performance of quantitative immunoassays is today largely restricted to centralized laboratories because of the need for long assay times, and relatively complex, bulky and expensive equipment, as well as highly trained operators. Most immunoassays remain within the walls of large centralized laboratories, far from the patients whose samples are collected and measured. If a wider range of the immunoassays are able to be run in a simpler way, less expensively and at the point of care or in the home health care environment, the health of many patients may be improved. To achieve this objective, a simple, compact, smart, robust, and inexpensive device providing high quality results is required.
Optical biosensors have some advantages, such as sensitivity, simplicity and immunity to electromagnetic wave interferences. Due to these advantages, optical biosensors are one type of biosensor exploited for immunoassay applications. There are many types of optical techniques which are commonly used for biosensing applications. Fluorescence-based sensors are highly developed due to their high sensitivity, versatility, accuracy and fairly good selectivity. A fluorescence method is also very suitable for miniaturization. The current technology to measure/detect fluorescent samples on a substrate such as, for example, inside a microfluidic channel, is performed by focusing the excitation light source onto the sample inside the microchannel and collecting the fluorescence emission of the sample using a set of complex lenses, mirrors, and optical filters. As a result, the fluorescence signal of microfluidic substrates may enter the detection system giving rise to a strong but unwanted fluorescence noise. The fluorescence from the sample of interest is usually very weak due to the low sample concentration. As a consequence, fluorescence noise due to the fluorescence of substrate may suppress the desired fluorescence signal from the sample of interest.
Currently, there are two approaches commonly used to avoid the noise due to the fluorescence of the substrate. The first approach is to incorporate a confocal fluorescence microscope to block the signals not from the thin layer within which the sample resides. This technique requires bulky, expensive and complicated optics. In the second approach, materials are selected with the substrate material having no, or a low, fluorescence property. Optical grade glass and silica are commonly used, since these materials do not fluoresce when they are excited by light within the visible wavelength range. However, these materials are relatively expensive and fabrication of microfluidic channels using these materials requires time-consuming photomask generation, photolithography and etching processes. As a consequence, a microfluidic chip made from optical grade glass or silica is relatively expensive.
Possible inexpensive materials suitable as substrate materials are polymer-based materials, such as polymethyl-methacrylate (PMMA), polycarbonate and Mylar. In addition, microfluidic channels using polymeric materials are easily fabricated by molding, embossing, casting or ablation processes. Complex models of microchannels in polymer sheets can be fabricated in less than an hour using a direct-write laser system. However, these materials exhibit relatively high fluorescence signals which in turn hinder their use for low fluorescence intensity detection. The intensity of the fluorescence background signal from the polymeric materials may be two orders of magnitude higher than the fluorescence signal of a sample within the microfluidic channel. Hence, there is a need to address the auto-fluorescence background noise of polymeric materials used in a polymeric microfluidic chip.